Establishment and application of a rapid and visual detection method for Mycoplasma pneumoniae using enzymatic recombinase amplification combined with lateral flow dipstick.

Mycoplasma pneumoniae (MP) is a key pathogen of respiratory tract infections, and the development of rapid, sensitive, and simple point-of-care testing (POCT) methods is of great clinical significance. In this study, enzymatic recombinase amplification (ERA) was combined with a colloidal gold-based lateral flow dipstick (LFD) to establish a novel MP detection method. Specific primers targeting the P1 protein gene were designed, with the downstream primer and probe labeled with biotin and 6-carboxyfluorescein (FAM) at their 5' ends, respectively, and the primer pair combinations, reaction temperature, and time were optimized to construct the ERA-LFD method. The detection performance for different MP strains was evaluated using reference samples and compared with a commercial fluorescent quantitative polymerase chain reaction (qPCR) kit. Subsequently, specificity, sensitivity, and clinical applicability were assessed using clinical specimens. The results showed that the target gene fragment of MP could be successfully amplified at 37 °C within 15 min, and the ERA amplification products could be visualized on the colloidal gold LFD by the naked eye. The limit of detection (LoD) of the assay was as low as 10 copies/μL, which was 1,000-fold more sensitive than conventional PCR, with no cross-reactivity to nucleic acids from other common respiratory pathogens. Evaluation of 158 clinical samples using qPCR as the gold standard demonstrated that the method achieved 98.0% sensitivity, 100.0% specificity, 98.7% overall agreement, and a Kappa value of 0.973. Collectively, this study developed a simple, rapid, and sensitive MP detection method that is suitable for both routine clinical applications and POCT scenarios.